Over the past years, a wide variety of specific therapeutic proteins, including antibodies, antibody fragments, and ligands for cell surface receptors have been developed and clinically tested. Exemplary proteins are antibodies, Fc-region conjugates, or targeted delivery vehicles. Some of these therapeutic proteins have been conjugated to several classes of therapeutic toxins such as small molecule drugs, enzymes, radioisotopes, protein toxins, and other toxins for specific delivery to patients.
Effective delivery to the site of disease is a prerequisite for high efficacy and low toxicity of any therapeutic molecule. For example, antibodies can participate in this context. If the antibody is not the therapeutic principle by itself, conjugation of an effector molecule to an antibody makes it possible to achieve precise localization of the drug at the desired site within the human body. This increases the effective drug concentration within this target area, thereby optimizing the therapeutic efficacy of the agent. Furthermore, with targeted delivery, the clinician may be able to lower the overall dose of the therapeutic agent and, thus, minimize systemic exposure—something that is particularly relevant if the drug payload has associated toxicities or if it is to be used in the treatment of chronic conditions (see e.g. McCarron, P. A., et al., Mol. Interventions 5 (2005) 368-380).
In WO 2010087994 methods for ligation and uses thereof are reported. Recombinant approaches to IgG-like bispecific antibodies are reported by Marvin, J. S., et al. (Acta Pharmacol. Sinica 26 (2005) 649-658). Levary, D. A., et al. (PLoS one, 6 (2011) e18342.1-e18342.6) report protein-protein fusion catalyzed by sortase A. In WO 2013/003555 the use of sortases to install click chemistry handles for protein ligation is reported.
Strijbis, K. et al (Traffic 13 (2012) 780-789) report protein ligation in living cells using sortase. It has been stated by them that the Ca2+-dependent S. aureus sortase A is not functional intracellularly, but that the Ca2+-independent S. pyogenes sortase A is functional in the cytosol and endoplasmic reticulum (ER) lumen of both Saccharomyces cerevisiae and mammalian HEK293T cells.